Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH3 and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2β activity, which is sensitive to wortmannin (10nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca2+ ionophore A23187 and blocked by the cell-penetrant Ca2+ chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2β revealed a single immunoreactive band of 180kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15min) exposure toμ-calpain, a similar gel shift together with an increase in PI3K-C2β activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2β activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and PI3K primarily in BLM, which leads to calpain-mediated activation of PI3K-C2β in BBM with a concomitant increase in PtdIns3P.
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August 2002
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Research Article|
August 01 2002
Hepatocyte growth factor activates phosphoinositide 3-kinase C2β in renal brush-border plasma membranes
Vladiana CRLJEN;
Vladiana CRLJEN
∗Department of Physiology and Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia
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Stefano VOLINIA;
Stefano VOLINIA
†Dipartimento di Morfologia ed Embriologia, Università degli Studi, Via Fossato di Mortara 64/b, 44100 Ferrara, Italy
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Hrvoje BANFIC
Hrvoje BANFIC
1
∗Department of Physiology and Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia
1To whom correspondence should be addressed, at Zavod za fiziologiju, Medicinski fakultet, Sveučilište u Zagrebu, Šalata 3, POB 978, 10001 Zagreb, Croatia (e-mail hrvoje.banfic@zg.tel.hr).
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Biochem J (2002) 365 (3): 791–799.
Article history
Received:
February 21 2002
Revision Received:
March 27 2002
Accepted:
April 03 2002
Citation
Vladiana CRLJEN, Stefano VOLINIA, Hrvoje BANFIC; Hepatocyte growth factor activates phosphoinositide 3-kinase C2β in renal brush-border plasma membranes. Biochem J 1 August 2002; 365 (3): 791–799. doi: https://doi.org/10.1042/bj20020316
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